The present invention is generally related to liquid handling systems for dispensing and aspirating small volumes of liquids of the order of 1 ml and smaller and in particular of the order of 50 microliters and smaller. Liquid handling systems are being provided and will be provided in the future for the dispensation of droplets of even smaller sizes. The present invention is particularly directed to liquid handling systems used in life science, medical and pharmaceutical sectors for applications such as high throughput screening, microarraying, Polymeraze Chain Reaction (PCR), combinatorial chemistry, proteomics, protein crystallography, genetic screening and others. The invention may also be used for medical diagnostics e.g. for printing reagents on a substrate covered with bodily fluids or for printing bodily fluids on substrates. The technology may also be used for applications outside the life science, medical and pharmaceutical sectors. For example it may be used for dispensing small volumes of lubricants for precision micromachining and drilling, and also for dispensing of small volumes of adhesives for the microelectronics industry and micromechanics and other applications.
The development of instrumentation for dispensing minute volumes of liquids has been an important area of technological progress for some time. Numerous devices for dispensing of small volumes of liquids of the order of 50 microliters and smaller have been developed over the past twenty-five years.
The requirements of a dispensing system vary significantly depending on the application. For example the main requirement of a dispensing system for ink jet applications is to deliver a droplet of a fixed volume with a high repetition rate. The separation between individual nozzles making up the ink jet dispenser should be as small as possible so that a number of nozzles may be accommodated on a single printing cartridge. On the other hand the task is simplified by the fact that the properties of the liquid dispensed, namely ink, are well defined and consistent.
For life science, medical and pharmaceutical applications the requirements are completely different. The system should be capable of handling a variety of liquids with different mechanical properties, e.g. viscosity. For many such applications it is important to be able to freely adjust the volume dispensed. Recent inventions related to the field of small volume liquid handling are covered by numerous patent applications, e.g. U.S. Pat. No. 5,744,099 (Chase et al); U.S. Pat. No. 4,574,850 (Davis); U.S. Pat. No. 5,035,150 (Tomkins), U.S. Pat. No. 5,741,554 (Tisone); U.S. patent application Ser. No. 09/709,541 (Shvets et al) filed 13 Nov., 1990, PCT patent Application No. /IE02/00039 (Shvets et al), filed 26 Mar. 2002, U.S. patent application Ser. No. 09/816,326 (Shvets et al) filed Mar. 26, 2001 and also described in scientific and technical publications [I. Schneider, Nanoliter Dispensing, Drug Discovery and Development, June 2002, p 51-54;
J. Comley, Nanoliter Dispensing—on the Point of Delivery, Drug Discovery World, Summer 2002, p 33-44;
D. Rose, Microdispensing Technologies in Drug Discovery, Drug Discovery Technology, vol 4, N9, September 1999, p 411-419;
S. D. Rose, Applications of a Novel Microarraying System in Genomics Research and Drug Discovery, Journal of the Association for Laboratory Automation, vol 3, N3, 1998, p 53-56;
I. V. Shvets, S. Makarov, C. Franken, A. Shvets, D. Sweney, J. Osing, Spot on Technology of low volume liquid Handling, Journal of Association of Laboratory Automation, vol 7, N 6, December 2002, p 127-131].
The wide variety of the mechanical properties of liquids and the very nature of many biological liquids often make consistent dispensing difficult. For example the dispenser can be easily blocked by a cloth as these are readily formed in cell-based liquids and protein solutions. Therefore, it is highly desirable to be able to verify that the instrument is dispensing correctly and in many cases to be able to detect the moment when the dispenser runs out of the liquid. It is also desirable to be able to detect if the liquid handling instrument leaks or if a drop fails to separate from the dispensing nozzle of the instrument. Additionally, in many instances, it is advantageous to be able to measure the volume of the droplet dispensed to ensure that it does correspond to the amount requested by the operator of the dispensing system. Independent verification of the volume dispensed becomes more and more important as the users have to operate in an environment of increasing legal regulation. For example, failure to dispense a drop may lead to an incorrect result of a medical test and consequently to incorrect diagnosis. Therefore, the user requirement for availability of such measurement technologies for operation verification intensifies.
It is also desirable for manufacturers of various liquid handling instruments to have in-house instrument test and calibration tools. During the production phase and also during the phase of development of new liquid handling instruments, it is important to have tools for quality control, calibration, tuning and optimisation of the instruments.
The issue of droplet volume measurement for small volume dispensing is also important from a psychological point. The reason is that in many cases the operator cannot readily monitor visually arrival of a tiny drop to a destination substrate, in particular, if the destination substrate is not flat. This adds to the user discomfort even if the dispenser functions properly as the user cannot readily satisfy himself/herself in this by simple visual monitoring.
It is difficult to fulfil challenging requirements for a successful system for measurement of droplet volume during dispensing. For many applications the ideal system must measure the volume in non-contact mode meaning that direct transfer of the drop to a measurement device such as microbalance is not an option.
U.S. Pat. No. 5,559,339 (Domanik) teaches a method for verifying a dispensing of a liquid droplet of relatively large size from a nozzle. The method is based on coupling of light from a source to a receiver. As a droplet of liquid travels from the nozzle it obstructs the coupling and therefore, the intensity of the signal detected by the receiver is reduced. The disadvantage of this method is that it is based on the absorption of electromagnetic radiation (in practice light) by the droplet. It will therefore not work for liquids that do no absorb the radiation well. For a range of applications where minute droplets of liquids with a broad range of optical properties need to be dispensed, the method may be inappropriate. Those familiar with the fundamentals of scattering of electromagnetic radiation by an object will readily appreciate that in order for this method to work, the wavelength of the electromagnetic radiation needs to be smaller than the droplet size.
Another method of volume measurement is based on detection of a charge carried by a droplet. The droplets are typically charged by applying a high voltage to the dispenser. The charge carried by the droplet depends on the droplet's size. In some inventions it is proposed using a Faraday pail for this purpose (U.S. patent application Ser. No. 09/709,541, filed 13 Nov., 1990 (Shvets et al); PCT/IE02/00039, filed 26 Mar. 2002 (Shvets et al); U.S. patent application Ser. No. 09/816,326 filed Mar. 26, 2001 (Shvets et al)). Faraday pails are well known and described in many published documents (see for example, Industrial Electronics by D. M. Taylor and P. E. Secker, Research Studies Press, 1994 ISBN) 0-471-1523333-8 and Electrostatics: Principles, Problems and Applications by J. Cross, A. Hilger ISBN 0-85274-589-3). Essentially the Farad The shield and the box are well insulated from each other. In this situation a charged droplet arriving at the box induces a charge of the opposite sign and same magnitude at the surface of the box. This charge is created by the current flowing to the inner box and it can be measured by a charge measurement circuit. Generally the dispenser and hence the nozzle are maintained at a relatively high voltage. The shield and box are connected to ground potential. The charge can also be measured without catching the droplet in the pail. For this the pail is made in the shape of a cylinder without a bottom. Thus the charged droplets progress through the Faraday pail that serves as an induced charge detector. The Faraday pail can be used to detect the moment when the droplet enters into the box and leaves it. Therefore, it can be used to measure the droplet's velocity, as the length of the box is known.
The Faraday pail has a number of disadvantages. One of the disadvantages is that it is sensitive to external electromagnetic noise, e.g. noise at 50 or 60 Hz frequency. To measure the volumes of small drops, the sensitivity of the Faraday pail must be optimised meaning that the entry and exit holes of the shield and the inner box must be reduced in size. This increases the chances of missing the exit hole and thus leaving the drop attached to the wall of the inner chamber. Another disadvantage is that in order to increase the charge carried by the droplet and thus improve the sensitivity of the instrument, it is often necessary to apply as high a voltage to the dispenser as possible. This may create further complications; e.g. the risk of destroying a charge sensitive amplifier connected to the pail, the risk of the malfunctioning of high voltage equipment with consequent danger for the operator, etc.
In some cases there is further complication related to the size of the drop. The liquid is often ejected from a dispenser in the form of a jet. If the total volume of the jet is small enough, the jet can then form a single drop as it travels through the air. This happens under the influence of the surface tension that favours the spherical shape over the cylindrical one. If the volume of the jet is rather large, the surface tension can alternatively split the jet into a number of drops. The length of the jet segment could be considerable even for relatively small volume of liquid ejected from the dispenser. For example, a 200 nl volume ejected from a nozzle with the diameter of 0.152 mm turns into a 11 mm long jet segment. This could be comparable to the size of the pail and therefore add to inaccuracy of the drop volume measurement as the jet segment can no longer be considered as a small charge surrounded from all the sides by the inner chamber of the Faraday pail.
To summarise, at present the issue of reliable detection and measurement of the volume of droplets dispensed is still not entirely resolved. It is suggested that the lack of a commonly acceptable measurement technology impedes wider use of low-volume liquid handling equipment.